Chemotherapy of microbial diseases 殷明

Shanghai Jiao Tong University 1940 年前，小儿肺炎、痢疾、伤寒、斑疹伤 寒、鼠疫、霍乱、白喉、疟疾 —— 不治之症 上海传染病死亡率 565/10 万 /10 万 1949 年，中国人平均寿命 39 岁 2004 年，中国人平均寿命 72 岁（日本 82 岁） 一些事实数据

Shanghai Jiao Tong University GENERAL PRINCIPLES OF ANTIMICROBIAL THERAPY

Shanghai Jiao Tong University Introduction Mechanism of action Antibiotic Resistance General principles of clinical use

Shanghai Jiao Tong University Definition In the strictest sense, antibiotics are antibacterial substances produced by various species of microorganisms (bacteria, fungi, and actinomycetes) that suppress the growth of other microorganisms. Common usage often extends the term antibiotics to include synthetic antimicrobial agents, such as sulfonamides and quinolones.

Shanghai Jiao Tong University classification according to the actions against the type of organism Antibacterial Antiviral Antifungal

Shanghai Jiao Tong University 1928: Alexander Fleming discovered the first antibiotic. He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus. Two Oxford University specialists Dr Howard W Florey and his German associate Dr Ernest Boris Chain spent month after month attempting to extract the essential substance from Fleming's mould. On 12 February, 1941, the first dose of isolated penicillin was injected into a human being Nobel Prize for Medicine in 1945 The discovery of Penicillin

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Antibiotics  Most significant discovery of modern medicine  Save millions of lives

Shanghai Jiao Tong University Academic terms Antimicrobial spectrum Bactericidal-MBC, bacteriostatic-MIC Therapeutic index : LD 50 /ED 50 Post antibiotic effect

Shanghai Jiao Tong University Concept 1: antibiotic spectrum Different antibiotics target different kinds of bacteria Antimicrobial spectrum Antimicrobial spectrum of a drug means the species of microorganisms that the drug can inhibit or kill. Narrow spectrum The agents act against a single or limited group of microorganisms (isoniazid: mycobacteria ) Extended spectrum The agent that are effective against gram-positive organisms and also against a number of gram- negative bacteria (ampicillin). Broad spectrum The agent affect a wide variety of microbial species (tetracycline)

Shanghai Jiao Tong University Concept 2: Bacteriostatic vs. Bactericide Antibiotics differ by mode of action Bacteriostatic: arrest the growth or replication of the microorganism. Sulfanilamide Bactericide: The agents which can kill the microorganisms are called bactericidal drugs; a drug may be bacteriostatic for one organism but bactericidal for another. Fluoroquinolones, Penicillins, …

Shanghai Jiao Tong University Concept 3: Bactericidal-MBC, bacteriostatic-MIC Chemotherapy index (CI) To evaluate the safety of chemotherapeutic drugs, the value is LD 50 /ED 50 Minimal inhibitory concentration (MIC) MIC is the lowest concentration of antimicrobial agents that prevents visible growth in hours incubation.

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Concept 4: Post antibiotic effect The antibiotic inhibits microbial multiplication beyond the time when the MIC is reached in plasma. Aminoglycosides Macrolides Allows reduction in dosing schedule

Shanghai Jiao Tong University Mechanism of action

Shanghai Jiao Tong University Inhibitors of cell wall synthesis Agents that interfere with cell membrane permeability Inhibitors of nucleic acid synthesis or activity Inhibitors of protein synthesis Inhibitors of bacterial metabolism … CLASSES OF ANTIBIOTICS

Shanghai Jiao Tong University Antimicrobial site of action

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Cell Wall Synthesis Inhibitors Penicillins 青霉素 Cephalosporins 头孢菌素 Vancomycin 万古霉素 Bacitracin 杆菌肽 β-lactam antibiotics

Shanghai Jiao Tong University Peptidoglycan （肽聚糖） is a 3D molecule Cross links are both horizontal and vertical between glycan chains stacked atop one another.

Shanghai Jiao Tong University Gram negative cell structure

Shanghai Jiao Tong University Antimicrobials acting on the bacterial cell wall Interfere with synthesis of peptidoglycan layer in cell wall eventually cause cell lysis bind to and inhibit activity of enzymes responsible for peptidoglycan synthesis “penicillin-binding proteins”(transpeptidase)

Shanghai Jiao Tong University β-Lactams 内酰胺 β-lactams inhibit transpeptidase. Only effective against rapidly growing organisms that synthesize peptidoglycan. The size, charge and hydrophobicity of the molecule determines the extent of its antibacterial activity.

Shanghai Jiao Tong University There is no molecule similar to peptidoglycan in humans, making drugs that target cell wall synthesis very selective in their toxicity against bacteria.

Shanghai Jiao Tong University Antimicrobials acting on the cell membrane amphotericin 两性霉素 : binding to the sterol- containing membranes of fungi polymyxins 多粘菌素 : acting like detergents and disrupt the Gram negative outer membrane. Not used parenterally because of toxicity to mammalian cell membrane fluconazole and itraconazole: interfering with the biosynthesis of sterol in fungi

Shanghai Jiao Tong University Bacteriostatic inhibitors of protein synthesis Board Spectrum: tetracyclines 四环素, chloramphenicol 氯霉素 Moderate: macrolides 大环内酯 Narrow: clindamycin 克林霉素 peroperties Suppress bacterial growth and replication but do not kill Second-line agents

Shanghai Jiao Tong University Antimicrobials acting on protein synthesis Target the bacterial ribosome. Bacterial – 70S (50S/30S) Mammalian – 80S (60S/40S) High levels may interact with mammalian ribosomes. Binding to 30s Subunit Aminoglycosides 氨基糖苷 (bacteriocidal) tetracyclines 四环素 Binding to the 50s subunit chloramphenicol 氯霉素 macrolides (erythromycin 红霉 素, clarithromycin 克拉霉素, azithromycin 阿齐霉素 )

Shanghai Jiao Tong University Antimicrobials acting on nucleic acid synthesis Inhibitors of DNA replication Quinolones (e.g. ofloxacin ，氧氟沙星 ) inhibit DNA-gyrase 回旋酶 Orally active, broad spectrum Damage to DNA Metronidazole 甲硝唑 (anti-anaerobes) nitrofurantoin 呋喃妥因 Inhibitors of Transcription rifampicin (key anti-TB drug) inhibits bacterial RNA polymerase flucytosine 氟胞嘧啶， incorporated into yeast mRNA

Shanghai Jiao Tong University Antimicrobials acting on Folic acid synthesis Inhibitors of precursor synthesis sulphonamides & trimethoprim are synthetic, bacteriostatic agents used in combination in co-trimoxazole （复方磺 胺甲噁唑 ） Sulphonamides inhibit early stages of folate synthesis （二氢叶酸合成酶） dapsone( 氨苯砜 ), an anti-leprosy drug Trimethoprim inhibits final enzyme in pathway, dihydrofolate reductase （二氢叶酸合成酶） Pyrimethamine （乙胺嘧啶）

Shanghai Jiao Tong University Mechanism of sulfonamides 蝶啶＋ → 二氢蝶酸 → 二氢叶酸 → 四氢叶酸 ↑ ↑ 二氢蝶酸合成酶 二氢叶酸还原酶 sulfonamides TMP （ 甲氧苄啶 ） Dapsone 氨苯砜 pyrimethamine （乙胺嘧啶） methotrexate

Shanghai Jiao Tong University Antibiotic Resistance

Shanghai Jiao Tong University 2010 年英国媒体：南亚发现新型超级病菌，产生 NDM-1 （ New Delhi metallo-β-lactamase-1 ， 新德里金属 -β- 内酰胺酶 -1 ），抗药性极强。 Superbug ： Escherichia coli and Klebsiella pneumoniae 克雷伯肺炎杆菌

Shanghai Jiao Tong University 日本科学家拍摄到的 “ 超级细菌 ” 照片

Shanghai Jiao Tong University Bacteria Antibiotic resistance  Definition: a microorganism is able to survive exposure to an antibiotic.  1940’s, penicillin-resistant bacteria, 1%  21st century, 耐苯唑西林金黄色葡萄球菌 （ MRSA: methicillin-resistant Staphylococcus aureus): 80-92%  耐万古霉素肠球菌（ VRE ），耐氨基糖苷类的 高耐株（ high-level aminoglycoside resistant, HLAR ）： 0-8% ， 60-80%  : 美国 center for disease control (CDC) 公布第一株真正的耐万古霉素（ MIC>128 mg/L ） 的葡萄球菌

Shanghai Jiao Tong University 金黄色葡萄球菌：获得性 耐药株的新挑战 39 th ICAAC 耐青霉素的金黄色葡萄球菌 ？ 古典的 MRSA 多重耐药 MRSA VRSA VISA VISA: Vancomycin-intermediate Staphylococcus aureus 万古霉素 中介耐药葡萄球菌 VRSA: vancomycin-resistant Staphylococcus aureus

Shanghai Jiao Tong University antibiotic era 抗生素时代 form 1940s: Penicillin was tested clinically and mass produced to 1975 Post-antibiotic era 后抗生素时代 multi-drug resistance 多药耐药

Shanghai Jiao Tong University The kinds of antibiotic resistance (1) Intrinsic resistance （天然 / 固有耐药）： genes in chromosome (2) Acquired resistance （获得性耐药） : genes in plasma transfer of antibiotic resistance:  transformation 转化  conjugation 接合  transduction 转导

Shanghai Jiao Tong University Mims C et al. Medical Microbiology

Shanghai Jiao Tong University mechanisms of resistance Inactivating enzymes: hydrolases; synthases Targets changes: protein structure change; production of protective protein Decrease of antibiotics: increased efflux system; decreased permeability to antibiotics Change of metabolic pathways; trapping mechanism; formation of biofilm

Shanghai Jiao Tong University Resistance to antibiotics

Shanghai Jiao Tong University Mechanisms of resistance Modification or bypass of target by mutation or acquisition of extrinsic DNA

Shanghai Jiao Tong University Resistance Mechanisms β-lactamase – hydrolyze the β-lactam ring. Penicillinase – Staph Alteration of penicillin-binding protein (PBP) affinity. (Strep. Pneumo., MRSA)

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 75% of antibiotics prescribed for respiratory tract infections  50% of antibiotic prescriptions are inappropriate  Most respiratory tract infections are caused by viruses  Antibiotics do not work against viruses Antibiotics in the Community

Shanghai Jiao Tong University Antimicrobial selection Identify the infecting organism Drug sensitivity of the infecting organism Host factors – site of infection, status of patient, safety of the agent, cost therapy

Shanghai Jiao Tong University Delaying the emergence of resistance Prescribed only when needed Narrow-spectrum: Cultures must be obtained prior to initiation of therapy Adequate dosage Limit use of newer drugs Minimize giving antibiotics to livestock

Shanghai Jiao Tong University Antibiotic combinations Severe infection Mixed infections Prevention of resistance – tuberculosis Decreased toxicity Enhanced antibacterial action

Shanghai Jiao Tong University Appropriate prophylactic use Before surgery Prevent bacterial endocarditis Protect fetus of an HIV-infected pregnant woman Prevent TB

Shanghai Jiao Tong University Quinolones, sulfonamides and other synthetic antibacterial drugs

Shanghai Jiao Tong University Basic structure of quinolones

Shanghai Jiao Tong University Quinolones （喹诺酮类） 1962 ， nalidixic acid （萘啶酸） pipemidic acid （吡哌酸） only used in urinary and intestinal infections caused by G － bacteria. low efficacy; rapid resistance. removed from clinical uses. Third and fourth generations: 1980’s: 氟喹诺酮类， norfloxacin （诺氟沙星）, ciprofloxacin （环丙沙星）， ofloxacin （氧氟沙星）， moxifloxacin （莫西沙星）， gatifloxacin （加替沙星）， sparfloxacin （司帕沙星） … (me-too)

Shanghai Jiao Tong University Mechanism of action Quinolones inhibit the bacterial DNA gyrase (topoisomerase Ⅱ ) or topoisomerase Ⅳ，拓朴异构酶Ⅳ ), thereby inhibiting DNA replication and transcription.

Shanghai Jiao Tong University Gyrase 能在两条链上都产生切口，然后重新连结， 并催化双链 DNA 形成双螺旋

Shanghai Jiao Tong University Resistance to quinolones seen in numerous pathogens, eg. Staphylococccus aureus （金黄色葡萄球菌）, enterococci （肠球菌） and Streptococcus pyogenes （化脓性链球菌）, … Mostly caused by inappropriate use (up to 99%)

Shanghai Jiao Tong University Resistance to quinolones efflux pumps decrease intracellular quinolone concentration. In gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. mutations at key sites in DNA gyrase or topoisomerase Ⅳ can decrease their binding affinity to quinolones, decreasing the drug's effectiveness.

Shanghai Jiao Tong University Common properties of quinolones Broad antibacterial spectrum: gram-negative and positive bacteria Some effective to pseudomonas aeruginosa （铜绿假单胞菌，（从前：绿脓杆菌）， mycobacterium tuberculosis （结核分枝杆 菌）， mycoplasma （支原体）， chlamydia （衣原体）， anaerobic bacteria （厌氧菌） Good pharmacokinetic features Less and mild untoward effects, but arthropathy （关节病） often seen in children and phototoxicity

Shanghai Jiao Tong University 衣原体 厌氧菌

Shanghai Jiao Tong University Individual quinolones Levofloxacin （左氧氟沙星） : chiral molecule, more powerful and less toxic Sparfloxacin （司帕沙星）： wider antibacterial spectrum including M. tuboculosis Moxifloxacin （莫西沙星）： high efficacy in anaerobic bacteria Gatifloxacin （加替沙星）： wide antibacterial spectrum and low phototoxicity

Shanghai Jiao Tong University sulfonamides （磺胺类） 对氨基苯磺酰胺

Shanghai Jiao Tong University Prontosil （百浪多息）

Shanghai Jiao Tong University History Gerhard Domagk ， German biochemist Involved in WW I, experienced lots of infections After MD, in dye company Paid attention to effects of dyes on bacteria Prontosil tested in mouse model of Streptococcus pyogenes infection Used in his daughter suffered from Streptococcus pyogenes infection and succeded Confirmed effective in President Roosevelt’s son 1939 Nobel prize in Medicine or Physiology

Shanghai Jiao Tong University Antibacterial spectrum of sulfonamides Antibacterial spectrum wide (bacteriostatic), but most of the bacteria resistant to these drugs now

Shanghai Jiao Tong University Mechanism of sulfonamides 蝶啶＋ → 二氢蝶酸 → 二氢叶酸 → 四氢叶酸 ↑ 二氢蝶酸合成酶 二氢叶酸还原酶 sulfonamides TMP （ 甲氧苄啶 ） Dapsone 氨苯砜 pyrimethamine （乙胺嘧啶） methotrexate

Shanghai Jiao Tong University Individual sulfonamides Sulfamethoxazole （磺胺甲噁唑） +trimethoprim （甲氧苄啶）＝复方新诺明； used in mild urinary or respiratory infections Sulfasalazine （柳氮磺吡啶）： ulcerative colitis （溃疡性结肠炎） Silver sulfadiazine （磺胺嘧啶银）： local use in burn wound

Shanghai Jiao Tong University nitrofurans （硝基呋喃类） Nitrofurantoin （呋喃妥因） urinary infections Furazolidone （呋喃唑酮） bacteroidal diarrhea